cDNA microarray analysis was used to examine gene expression profiles in chemically-induced rat mammary gland carcinomas. Several genes showed increased expression in carcinomas by cDNA microarray analysis and were further validated by immunohistochemistry and western blot analysis. These genes were cyclin D1, PDGF-A chain, retinol binding protein 1, prohibitin, and the transcription factor Stat5a. Recently, this laboratory examined the expression of signal transducer and activator of transcription (Stat) 5a in rat mammary gland carcinomas and in human breast cancers. Stat5a expression was examined immunohistochemically in rat mammary gland carcinomas induced by the chemical carcinogens 7,12-dimethylbenzaanthracene (DMBA)and 2-amino-1-methyl-6-phenylimidazo4,5-bpyridine (PhIP). A high percentage of carcinomas showed nuclear labeling of Stat5a 44 of 68 (65%) with Stat5a nuclear labeling index ranging from 18-77%. In contrast, control normal mammary gland tissue displayed cytosolic expression. Carcinomas with different Stat5a staining patterns (cytoplasmic or nuclear) showed a statistical difference for the proliferating cell nuclear antigen (PCNA) labeling, tumor differentiation, nuclear grade, mitotic activity, and tumor size. High Stat5a nuclear expression was closely correlated with the higher-grade, more poorly differentiated carcinomas. Stat5a nuclear expression was also detected in the intraductal proliferation (10 of 21 lesions) and in ductal carcinoma in situ (13 of 15 lesions). Immunohistochemical analysis was further carried out in human breast cancers. Stat5a nuclear expression was detected in ductal and lobular carcinomas and DCIS at a frequency of 48% (15/31), 33% (2/6), and 40% (2/5), respectively. Nuclear expression of Stat5a in human breast cancers also correlated with PCNA nuclear labeling index. The findings implicate activated Stat5a in mammary gland cancer development in the rat and human. Further studies on an unknown clone initially identified through microarray analysis lead to the cloning an identification of rat Id4 (GenBank accession No. AF468681). Id4 regulates mammary epithelial cell growth and differentiation and is over-expressed in rat mammary gland carcinomas. Id4 belongs to a family of helix-loop-helix (HLH) proteins that impact cellular growth and differentiation via regulation of basic HLH transcription factors. The expression of rat Id4 was examined in rat mammary gland tumors induced by 2-amino-1-methyl-6-phenylimidazo4,5-bpyridine (PhIP), a carcinogen found in the human diet. By real-time polymerase chain reaction analysis, relative expression of Id4 mRNA in carcinomas, adenomas and normal tissue was 27, 6, and 1, respectively. Immunohistochemical analysis indicated statistically elevated nuclear expression for Id4 protein in carcinomas in comparison to adenomas and normal mammary gland. In carcinomas, Id4 nuclear expression was positively correlated with proliferation, invasiveness, and tumor weight (Fisher Exact Test or Spearman Correlation, p<0.05). The consequence of enforced expression of Id4 on mammary epithelial cell proliferation, differentiation, and growth in soft agar was examined in HC11 cells, a well-characterized model for studying various aspects of mammary epithelial cell biology. After transient and stable transfection of HC11 cells, Id4 over-expression increased cell proliferation and inhibited lactogenic hormone-mediated differentiation as revealed by inhibition of b-casein promoter activity and b-casein expression. In addition, enforced expression of Id4 in HC11 cells induced a statistically significant increase in colony growth in soft agar. The results implicate Id4 in rat mammary gland carcinogenesis and suggest that Id4 may contribute to carcinogenesis by inhibiting mammary epithelial cell differentiation and stimulating mammary epithelial cell growth.